Tissue-adhesive formulations

ABSTRACT

A tissue-adhesive formulation consists of a naturally occurring or synthetic polymerisable and/or cross-linkable material in particulate form, the polymerisable and/or cross-linkable material being in admixture with particulate material comprising tissue-reactive functional groups. The formulation may be used in the preparation of a tissue-adhesive sheet, by applying the formulation to at least one side of a core of a naturally occurring or synthetic polymeric material.

This is a national stage application under 35 U.S.C. § 371 ofPCT/GB2004/001464, filed Apr. 2, 2004, which claims the priority benefitof British patent applications GB 0307765.8 filed Apr. 4, 2003, GB0317447.1 filed Jul. 25, 2003, and GB 0318871.1 filed Aug. 12, 2003.

FIELD OF THE INVENTION

This invention relates to materials suitable for use as tissue adhesivesand sealants, and to a flexible multilamellar sheet, patch or filmcomprising such materials for topical application to internal andexternal surfaces of the body, for therapeutic purposes. The inventionalso relates to a process for the preparation of such products, and tomethods of using such products. In particular the invention relates tomaterials that are formulated as loose or compacted powders and to aself-adhesive, biocompatible and hydratable polymeric sheet with suchmaterials applied to a suitable support, which may be used fortherapeutic purposes such as wound healing, joining, sealing andreinforcing weakened tissue, and for drug delivery, and to a process forpreparing, and methods of using, such a sheet.

BACKGROUND OF THE INVENTION

There is considerable interest in the use, for a number of surgical orother therapeutic applications, of materials that adhere to biologicaltissues eg as an alternative to the use of mechanical fasteners such assutures, staples etc. Formulations of such materials that have hithertobeen proposed include viscous solutions or gels that are eithermanufactured in that form or are prepared immediately prior to use bymixing of the ingredients. Such formulations are then applied to thetissue surface using a suitable applicator device such as a syringe.

Formulations of the type described above suffer from a number ofdisadvantages. If the formulation is of low viscosity, it may spreadfrom the area of application and hence be difficult to apply preciselyto the desired area of tissue. If the formulation is more viscous, onthe other hand, it may be difficult to dispense. In either case, theformulation, being prepared in hydrated form, may have a limitedlifetime and may be subject to premature curing. It may therefore benecessary for the whole of the formulation to be used at once ordiscarded. Also, the preparation of formulations immediately prior touse by mixing of ingredients is obviously laborious and time-consuming.In addition to these drawbacks, the degree of adhesion between tissuesurfaces that is provided by such formulations may be less than would bedesired.

Formulations of tissue adhesive materials have also been applied to asuitable support for application to the tissue surface. The use oftherapeutic materials in the form of a sheet, patch or film, for topicaladministration to either internal or external organs of the body, iswell documented for a wide range of medical applications. A disadvantageof products proposed hitherto, however, is that the degree of adhesionto the underlying tissue, particularly in the longer term, may beinadequate. While the initial adhesion may be satisfactory, the sheetmay subsequently become detached from the tissue, often after only a fewseconds or minutes, eg as a result of hydration of the sheet followingits application. In addition, the flexibility of the product may beinsufficient for it to conform readily to the surface to which it isapplied, which may also have an adverse effect on its adhesion.

As a result of the inadequate adhesion of these products, it may benecessary to provide further reinforcement, eg through mechanicalattachment using sutures, staples or the like. Alternatively, energy (eglight or heat energy) may be applied in order to initiate chemicalbonding of the adhesive formulation to the underlying tissue, and hencebonding of the tissue surfaces to each other. Clearly, such approachesintroduce further drawbacks. The use of mechanical fastenings such assutures or staples is often the very thing that the use of such productsis intended to replace or avoid. In many instances, the use of suchfastenings is either not wholly effective (eg on the lung) orundesirable, as their introduction gives rise to further areas of tissueweakness. The use of external energy requires the provision andoperation of a source of such energy. Such energy sources may beexpensive and difficult to operate, particularly in the confines of anoperating theatre or similar environment. Also, the use of externalenergy for attachment can be both time-consuming and (in some cases)requires significant careful judgement on the part of the surgeon, toevaluate when sufficient energy has been delivered to effect attachmentwithout damaging the underlying tissue.

There have now been devised improved formulations of tissue-adhesivematerials and sheets or the like of the general type described abovethat overcome or substantially mitigate the above-mentioned and/or otherdisadvantages of the prior art.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided atissue-adhesive formulation comprising a naturally occurring orsynthetic polymerisable and/or cross-linkable material in particulateform, the polymerisable and/or cross-linkable material being inadmixture with particulate material comprising tissue-reactivefunctional groups.

The formulation according to the invention is advantageous primarily inthat it can be easily applied to a tissue surface using a simpleapplicator or delivery device. As it is applied in solid form, theparticulate formulation adheres to the tissue surface and does notspread unduly. The formulation exhibits good initial adhesion to thetissue surface, this being believed to be due to van der Waals forcesand/or hydrogen bonding between the formulation and the tissue surface.On contact with the tissue surface the formulation becomes hydrated,thereby causing reaction between the tissue-reactive functional groupsand the underlying tissue surface. Such reactions between thetissue-reactive functional groups and the underlying tissue results inhigh adhesion between the formulation and the tissue surface, and hencebetween tissues hat are joined using the adhesive formulation. Reactionmay also take place between the tissue-reactive functional groups andthe other components of the formulation to form a strong, flexible andtissue-adherent gel. This formulation thus absorbs physiological fluids(as a consequence of application onto exuding tissue surfaces), and anyadditional solutions used to hydrate the formulation followingapplication (such fluids can be commonly used solutions used in surgicalirrigation), becoming gelatinous and adherent to the tissue surfaces,and thereby providing an adhesive sealant, haemostatic and pneumostaticfunction.

In addition, because the formulation is made up in solid form that is,until hydrated by contact with the tissue surface (and subsequenthydration), essentially inactive, the formulation is not prone topremature reaction and as a result its shelf-life may be considerable,eg more than six months when stored at room temperature and storedappropriately. This further enables the formulation to be packaged inrelatively large quantities that can be dispensed and used over aconsiderable time period, without the risk of substantial wastage.

According to a second aspect of the present invention, there is provideda sheet having a multilayer structure, said structure comprising a coreof a naturally occurring or synthetic polymeric material, the core beingcoated on at least one side thereof with a tissue-adhesive formulationcomprising a naturally occurring or synthetic polymerisable and/orcross-linkable material in particulate form, the polymerisable and/orcross-linkable material being in admixture with particulate materialcomprising tissue-reactive functional groups.

In preferred embodiments of the invention, the tissue-adhesiveformulation is applied to the core by mechanically compressing a blendof material containing tissue-reactive functional groups (hereinafterreferred to as “tissue-reactive material”) and the polymerisable and/orcross-linkable component, both in particulate form, onto one or bothsides of the core.

The sheet according to the invention is advantageous primarily in thatit bonds effectively to tissue, enabling it to be used in a variety ofmedical applications. The invention enables coating of thetissue-reactive materials onto (and into) a three-dimensional structuralsupport, whilst maintaining the pliability and physical properties ofthe support. Furthermore, the adhesive performance of thetissue-reactive materials is not compromised when delivered to thetarget tissue in this form. Where, as in preferred embodiments, thesupport is perforated, the perforations provide a means of anchoring thetissue-reactive materials in the support. This reduces or eliminatescracking and crumbling of the tissue-reactive material as it is appliedto the support (core), which would result in sub-optimal coverage of thetarget tissue, and thereby compromise the adhesive/sealant effects ofthe sheet.

The sheet exhibits good initial adhesion to the tissue to which it isapplied (and may thus be described as “self-adhesive”), and furthermoreremains well-adhered to the tissue over a longer timescale. Withoutwishing to be bound by any theory, it is believed that the initialadhesion of the sheet to the tissue is attributable to electronicbonding of the sheet to the tissue, and this is supplemented or replacedby chemical bonding between the tissue-reactive functional groups of theformulation and the tissue, in particular between amine and/or thiolgroups on the tissue surface and the tissue-reactive groups of thesheet. Where the structural inner core of the device is perforated, andis coated on both sides with the tissue-adhesive formulation, theperforations facilitate hydration and cross-linking of the formulationon both sides of the core such that the core becomes enclosed within athree-dimensional matrix of cross-linked material.

The use of the sheet reduces or eliminates the need for additional meansof mechanical attachment to the tissue (eg sutures or staples), or theneed to provide external energy in the form of heat or light to bringabout adherence of the sheet to the underlying tissue. Another advantageof the sheet according to the invention is that it is applied to thetissue as a preformed article, rather than being prepared by mixing ofmaterials immediately prior to use.

By the term “sheet” is meant a three-dimensional article with athickness that is considerably less than its other dimensions. Such anarticle may alternatively be described as a patch or a film.

According to another aspect of the invention, there is provided a methodfor the manufacture of a sheet according to the second aspect of theinvention, which method comprises forming a core comprising naturallyoccurring or synthetic polymeric material, and coating at least one sideof said core with a tissue-adhesive formulation comprising a blend of anaturally occurring or synthetic polymerisable and/or cross-linkablematerial in particulate form and particulate material comprisingtissue-reactive functional groups.

In a third aspect, the invention also provides a method of joining atissue surface to another tissue, or of sealing a tissue surface, whichmethod comprises applying to the tissue surface a formulation accordingto the first aspect of the invention or a sheet according to the secondaspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the reaction between atissue-reactive functional group (in the illustrated case anN-hydroxysuccinimide ester) and an amine-containing molecule such as atissue protein.

FIG. 2 shows the introduction of carboxyl group-bearing side chains intoa poly(vinyl alcohol—vinyl acetate) copolymer.

FIG. 3 represents the formation of apoly(N-vinyl-2-pyrrolidone-co-acrylic acid) copolymer.

FIG. 4 shows the mechanism of free radical initiation of apolymerisation reaction.

FIG. 5 illustrates the synthesis of a tissue reactive material.

FIG. 6 is a schematic sectional view of a sheet according to theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Composition of the Formulation

The formulation according to the first aspect of the invention comprisesa naturally occurring or synthetic polymerisable and/or cross-linkablematerial in particulate form, and a particulate material comprisingtissue-reactive functional groups.

These two components may be blended in suitable proportions, which maydepend on the particular materials used, as well as the desiredproperties of the resulting blend. Typically, the ratio by weight ofpolymerisable and/or cross-linkable material to tissue-reactive materialwill be between 0.1:1 and 10:1, more preferably between 0.2:1 and 1:1.

The particles that make up the formulation have a wide range of particlesizes. The median particle size may, for instance, lie in the range 5 μmto 500 μm, more preferably 5 μm to 100 μm.

Nature of the Polymerisable and/or Cross-Linkable Component

One component of the formulation is a polymerisable and/orcross-linkable material.

By “polyerisable” is meant that the material may be present in theformulation as a prepolymer or macromer in monomeric or only partiallypolymerised form, such that upon hydration of the formulation thematerial undergoes (further) polymerisation.

More commonly, however, the material will be “cross-linkable”, ratherthan “polymerisable”. By this is meant that the material will be capableof forming covalent bonds between molecules. Such inter-molecularcross-linking may also be accompanied by intra-molecular cross-linking,ie formation of covalent bonds between functional groups in the samemolecule.

The cross-linkable material will generally be polymeric ormacromolecular in form, the effect of the cross-linking being to formcovalent bonds between such molecules, and so to establish athree-dimensional network or matrix.

The cross-linkable material is preferably selected from polysaccharides,polylactates, polyols and proteins, and derivatives thereof.

The cross-linkable material may be in a partially crosslinked form inwhich individual molecules of the cross-linkable material are linkedtogether through intermolecular covalent bonds. Such crosslinking can beeffected by standard techniques known in the art, for example by heattreatment and/or crosslinking agents. Depending on the nature of thecross-linkable material and/or the conditions employed to effectcrosslinking, the degree of crosslinking between individual moleculescan vary considerably.

The degree of pre-crosslinking in the cross-linkable material, however,should not be such that it substantially prevents the subsequentreaction of the tissue-reactive functional groups with thecross-linkable material.

Proteins are preferred materials for the cross-linkable material becausethey are rich in functional groups that are reactive to tissue-reactivefunctional groups. Hence, the tissue-reactive functional groups willreact not only with the tissue surface to which the formulation isapplied, but also with the cross-linkable material.

A particularly preferred protein for use in the invention is albumin,particularly mammalian albumin such as porcine, bovine or human albumin.

Preferred synthetic polymers that may be, or may be present in, thepolymerisable and/or cross-linkable material include multifunctionallyactivated synthetic polymers, ie synthetic polymers that have, or havebeen chemically modified to have, a plurality of functional groups thatare capable of reacting with each other or with other functional groupspresent in the formulation to form covalent bonds. Preferredmultifunctionally activated synthetic polymers include chemicallymodified polyalkylene glycols, in particular polyalkylene glycols thathave been modified to contain multiple primary amino or thiol groups.

Suitable modified polyalkylene glycols include both linear and branched(eg so-called “3-arm” and “4-arm”) compounds. Examples of suitablemulti-amino polymers are those sold under the trade name JEFFAMINE.These are based on backbones of polyethylene glycol and/or polypropyleneglycol units with terminal amine groups.

Other suitable synthetic materials may include, or may be based on,poly(vinylamine), poly(ethyleneimine), poly(allylamine), poly(ethyleneglycol-co-aspartic acid, poly(lysine-co-lactide,poly(cysteine-co-lactide) or poly(2-aminoethylmethacrylate).

In general, suitable cross-linkable materials will be materials that aresolid at ambient temperatures, and this may preclude the use of very lowmolecular weight materials.

Methods by which similar and analogous chemically modified polymers canbe prepared will be readily apparent to those skilled in the art.

Nature of the Tissue-Reactive Material

The tissue-reactive material is preferably polymeric in nature. Mostpreferably, the polymer is a synthetic polymer.

By “tissue-reactive functional groups” is meant functional groupscapable of reacting with other functional groups present in the tissuesurface so as to form covalent bonds between the formulation and thetissue. Tissues generally consist partly of proteins, which commonlycontain thiol and primary amine moieties. Many functional groups such asimido ester, p-nitrophenyl carbonate, N-hydroxysuccinimide (NHS) ester,epoxide, isocyanate, acrylate, vinyl sulfone, orthopyridyl-disulfide,maleimide, aldehyde, lodoacetamide, and others, may react with thiols orprimary amines, and therefore constitute “tissue-reactive functionalgroups”. As used herein, the term NHS or NHS ester is intended toencompass not only N-hydroxysuccinimide itself, but also derivativesthereof in which the succinimidyl ring is substituted. An example ofsuch a derivative is N-hydroxysulfosuccinimidyl and salts thereof,particularly the sodium salt, which may increase the solubility of thetissue-reactive material.

FIG. 1 illustrates the mechanism by which an NHS-functionalised polymerreacts with an amine-containing material such as a tissue proteinrepresented by R—NH₂. The reaction is a nucleophilic displacementleading to the formation of an amide bond between the polymer and thetissue protein.

Tissue-reactive functional groups that may be of utility in the presentinvention are any functional groups capable of reaction (under theconditions prevalent when the formulation is applied to tissue, ie in anaqueous environment and without the application of significant amountsof heat or other external energy) with functional groups present at thesurface of the tissue. The latter class of functional group includesthiol and amine groups, and tissue-reactive functional groups thereforeinclude groups reactive to thiol and/or amine groups. Examples are:

-   -   imido ester;    -   p-nitrophenyl carbonate;    -   N-hydroxysuccinimide (NHS) ester;    -   epoxide;    -   isocyanate;    -   acrylate;    -   vinyl sulfone;    -   orthopyridyl-disulfide;    -   maleimide;    -   aldehyde; and    -   iodoacetamide.

N-hydroxysuccinimide (NHS) ester is a particularly preferredtissue-reactive functional group.

In general, the tissue-reactive material may be formed by derivatisationof a suitable polymer precursor. Classes of polymer which lendthemselves to such derivatisation include those that contain carboxylicacid or alcohol functional groups, or related structures. Polymers thatmay be used include polymers that are commercially available or polymersthat are prepared specifically for this purpose. Naturally-occurringmaterials such as sucrose or a derivatised cellulose may also be used.

Commercially available polymers that may be used includepolyvinylalcohol (PVA). In the case of PVA, the functional groups may beintroduced by first adding a chain extending or linking group, forexample an acid functionality that can be further reacted with N-hydroxysuccinimide. FIG. 2 shows the addition of a chain-extending group to acopolymer of vinyl acetate and vinyl alcohol, the chain-extending groupterminating in a carboxylic acid group that may be converted to thecorresponding NHS-ester. The copolymer starting material (in which molarfraction x of vinyl alcohol groups may be 0.85-0.995) is reacted with acyclic anhydride (in the example illustrated, succinic anhydride) in thepresence of a base such as pyridine. Between 5% and 40% of the alcoholgroups are derivatised to form the carboxylic acid-bearing side chains(ie a+b=x, with a being between 0.05x and 0.40x), which may then beconverted to the NHS-ester by conventional methods that are known perse.

Where the polymer support is synthesized for the purpose of subsequentderivatization, a wide variety of monomers may be used. Examples includeN-vinyl-2-pyrrolidone, acrylic acid, vinyl acetate, vinyl acetic acid,mono-2-(methacryloyloxy)ethyl succinate, methacrylic acid,2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate, (polyethyleneglycol) methacrylate or other monomers containing acid or alcoholfunctionality. Such monomers may be polymerised via various standardpolymerisation techniques, including free radical techniques using aninitiator such as benzoyl peroxide, 2,2′-azobisisobutyronitrile (AIBN),lauroyl peroxide, peracetic acid etc. One preferred example of such apolymer is poly(N-vinyl-2-pyrrolidone-co-acrylic acid) polymerised usingAIBN as initiator. The polymerization of this material is illustrated inFIG. 3, in which the molar ratio of acrylic acid-derived units may bebetween 0 and 1.0, preferably less than 0.60, and more preferably lessthan 0.40, eg between 0.025 and 0.25. The copolymer may be furtherreacted with N-hydroxysuccinimide to form the tissue-reactive material.

Preferably, all or substantially all of the available sites in theprecursor to the tissue-reactive material will be derivatised (ie thetissue-reactive functional groups will be introduced into all orsubstantially all of the available sites in the precursor to thetissue-reactive material). The degree of binding between thetissue-reactive functional groups and the tissue to which theformulation is applied will then be a function of the amount oftissue-reactive material in the formulation.

Where, as is preferred, the tissue-reactive functional groups areNHS-esters, at least one of the monomers used in the preparation of thetissue-reactive material must contain a carboxylic acid group or a groupcapable of being reacted with another material to form an acidfunctionality.

In the preferred case in which the tissue-reactive material is aderivative of poly(N-vinyl-2-pyrrolidone-co-acrylic acid) copolymer(PVP-co-PAA), the molar ratio of acrylic acid-derived units ispreferably between 0.05 and 0.50, and hence that of the vinylpyrrolidone-derived units is between 0.50 and 0.95. The derivative isreferred to as activated PVP-co-PAA.

The acrylic acid groups are preferably derivatised to formtissue-reactive (activated) groups, most preferably with NHS groups. Acopolymer of vinyl pyrrolidone and acrylic acid, in which the carboxylgroups of the acrylic acid-derived units carry NHS groups, is referredto herein as NHS-activated PVP-co-PAA.

As noted above, the activity of the tissue-reactive material (ie thedegree to which the tissue-reactive functional groups of that materialbind to the tissue) may be controlled by varying the proportion of thatmaterial in the formulation. The concentration of the tissue-reactivematerial in the formulation may be varied quite widely, eg from 10% w/wor less up to 50% w/w or more.

The formulation may contain one type of tissue-reactive material, ormore than one type of tissue-reactive material.

In addition to forming covalent bonds with the surface to which it isapplied, the tissue-reactive material may also have bioadhesiveproperties. By this is meant that the material should exhibit goodinitial adhesion to biological tissue to which it is applied. Polymerswith such properties typically contain chemical groups with a high ionicdensity, eg carboxyl, amide, lactam, hydroxyl, ether and ester groups,and the salts thereof, which interact cooperatively with tissue, throughthe formation of ionic and hydrogen bonds, dipole—dipole interactionsand Van der Waals forces. Effective polymers are generally of highmolecular weight since the degree of bioadhesion may be proportional tothe number of these groups available. Typically, the molecular weight ofa bioadhesive polymer will be in excess of about 50,000. The polymersare also generally linear, becoming physically entangled and having anamorphous distribution in solution.

For example, the tissue-reactive polymer may be derivatised PVP or aderivatised copolymer of vinyl pyrrolidone with another monomer (egacrylic acid). In such a case, the pendant pyrrolidone groups willprovide immediate contact adhesion (believed to be due to hydrogenand/or van der Waals bonding, as described above), while thetissue-reactive groups then form covalent bonds with functional groupswithin the tissue and within the matrix (cross-linking).

Thus, according to another aspect of the invention there is provided abiocompatible and hydratable composition suitable for topicalapplication to internal or external surfaces of the body, which matrixcomprises a polymer containing tissue-reactive functional groups and apolymer containing groups that are not tissue-reactive functional groupsbut which are capable of forming hydrogen bonds with groups at thesurface of a tissue to which the matrix is applied.

The tissue-reactive functional groups may be as described above. Thegroups that are capable of forming hydrogen bonds (“hydrogen-bondinggroups”) are preferably electron-rich groups, eg selected from amide,lactam, carbonyl, carboxyl, hydroxyl and ether groups.

In a particular embodiment, the tissue-reactive groups and thehydrogen-bonding groups are present in the same polymer. The inventionthus provides a biocompatible and hydratable composition suitable fortopical application to internal or external surfaces of the body, whichcomposition comprises a polymer containing tissue-reactive functionalgroups and groups that are not tissue-reactive functional groups butwhich are capable of forming hydrogen bonds with groups at the surfaceof a tissue to which the matrix is applied.

The tissue-reactive groups are preferably tissue-reactive ester groups,especially NHS-ester groups, and the hydrogen-bonding groups arepreferably amide or lactam groups. The polymer is thus preferablyactivated PVP-co-PAA, especially NHS-activated PVP-co-PAA.

Alternatively, the tissue-reactive groups and the hydrogen-bondinggroups may be present in different polymers. In addition to thetissue-reactive material, therefore, the formulation according to theinvention may further comprise a polymer containing hydrogen-bondinggroups. Examples of suitable polymers are poly(vinylic acids) andcopolymers thereof. However, a preferred group of bioadhesive polymersare polymers consisting of a hydrocarbon backbone with pendant amide orlactam groups, or recurring structural units containing such groups.Preferably, the recurring unit is, or contains, a1-ethylenepyrrolidin-2-one (vinylpyrrolidone) group. Homopolymerscontaining recurring N-vinyl-2-pyrrolidone groups are particularlypreferred, ie poly(vinylpyrrolidone) (PVP).

PVP has been found to be suitable for use in the present invention fornumerous reasons. First, it is readily available in a variety of gradesand molecular weights, and has a long history of use in medicalapplications. PVP has a linear structure, is stable over wide ranges ofpH and temperature, and is readily and rapidly soluble in water andother solvents.

Without wishing to be bound by any particular theory, it is believedthat the good bioadhesive properties of PVP are attributable to the factthat the carbonyl group and nitrogen atom of the pendant pyrrolidonemoiety are electron-rich. The material is therefore capable ofeffectively immediate adhesion to the tissue to which it is appliedthrough hydrogen bonds formed with functional groups at the surface ofthe tissue and/or through van der Waals forces.

The bioadhesive polymer may alternatively be a copolymer, eg a copolymerof amide- or lactam-containing units as described above and a vinylicacid. One particular form of copolymer that may be suitable is thuspoly(vinylpyrrolidone-co-acrylic acid) (referred to herein asPVP-co-PAA).

Other groups of polymers that may exhibit suitable bioadhesiveproperties include cellulose derivatives, particularly cellulose ethersand derivatives and salts thereof. Examples include carboxymethylcellulose (CMC) and salts thereof, hydroxypropylmethyl cellulose andhydroxyethylmethyl cellulose. Sodium carboxymethyl cellulose is oneexample of such a polymer.

Combinations of polymers of the kinds described above may be employed.One preferred example is a combination of a polymer of amide- orlactam-containing units as described above and a cellulose derivative asdescribed above. A particular combination is PVP and a salt, eg thesodium salt, of CMC.

Sufficiency of the degree of initial adhesion of a sheet to the tissue,by the bioadhesive polymer(s), can be quantitatively determined invitro, for example by performing an adhesion strength test. This test isperformed by allowing the sheet to adhere to a suitable substrate(secured in a fixed position), while the sheet itself is physicallyattached at a separate point to the load of a tensile testing apparatus,positioned so that, prior to the test, the sheet is not under load. Theload cell is moveable along an axis substantially perpendicular to thatalong which the substrate is positioned. The test involves movement ofthe load cell away from the substrate, at a constant predetermined rate,until the sheet detaches from the substrate. The output of the test is aquantitative measure of the energy of adhesion for that sheet—ie thecumulative amount of energy required to break the interaction betweenthe sheet and the substrate to which it is adhered. A suitablecumulative energy of adhesion for the sheet according to the inventionwould be not less than 0.1 mJ, more preferably not less than 0.25 mJ,and most preferably not less than 0.5 mJ.

Manufacture of the Components of the Formulation

The particulate tissue-reactive material and the particulatepolymerisable and/or cross-linkable component may be prepared by anysuitable means. Particularly where the latter component isproteinaceous, the particles are preferably prepared by freeze- orheat-drying of an aqueous solution or suspension.

To enhance the reaction between the tissue-reactive material (which istypically an electrophile-rich material) and the polymerisable and/orcross-linkable material (which is typically nucleophilic), it may benecessary or desirable to buffer the latter component to alkaline levelsto promote proton extraction.

It should be apparent to those skilled in the art that the reactionbetween electrophilic and nucleophilic compounds may be controlled byadjusting the pH of the reaction. As the pH is adjusted above 7, thecrosslinking reaction becomes more favourable.

In preferred embodiments of the present invention, polymerisable and/orcross-linkable nucleophile-rich materials are processed by alkalinebuffering and subsequent lyophilisation. These materials aresubsequently mixed with tissue-reactive materials in a dehydrated form.Upon hydration (ie during application onto tissue surfaces) the alkalinebuffering of the formulation ensures effective reaction between thecrosslinkable material and the associated tissue-reactive material.

By using such a method, the need for the components of the formulationto be buffered separately immediately prior to use is eliminated. Thenovel method described herein eliminates the requirement to “prime” thetarget site or to prepare a range of buffered solutions in order toeffect a cross-linking reaction.

The degree of buffering required is dependent on the cross-linkablematerial being utilised and the conditions required to abstract a protontherefrom. For example, it has been found that for human albumin theoptimal pH required to ensure effective crosslinking is in the range pH9-11. For synthetic polymeric compounds such as polyvinylamine, on theother hand, equally effective reactivity is demonstrated at pH 7-8.

Thus, in preferred embodiments of the invention, the polymerisableand/or cross-linkable material is buffered to a pH greater than 7.

The formulation may be prepared simply by admixing the components inparticulate form, and where desired compacting the formulation to formtablets, plugs or the like. The degree of compaction should be such thatthe tablets etc retain their integrity until applied to tissue, but notso great as to inhibit hydration (and hence adhesion) after application.

Physical Forms of the Formulation

The formulation according to the invention may have the form of a loosepowder, in which particles of the tissue-reactive material are admixedwith particles of the polymerisable and/or cross-linkable component.

Alternatively, the formulation may take the form of a compacted bodyformed by compaction of the particles. Tissue-reactive materials basedon poly (N-vinyl-2-pyrrolidone) or copolymers of N-vinyl-2-pyrrolidonewith other monomers (eg vinylic monomers) are particularly preferred insuch applications, as poly (N-vinyl pyrrolidone) has suitable flowproperties for blending with other components of the formulation, andexhibits excellent performance in dry granulation tableting processes asit undergoes plastic deformation on compression, and has lowhygroscopicity.

In a further alternative, the formulation may be applied to a core toform a sheet according to the second aspect of the invention.

Sheets in accordance with the invention may be planar, or may be folded,fluted, coiled or otherwise formed into more complex shapes.

The formulation may further comprise additional components such asstructural polymers, surfactants, plasticizers and excipients commonlyused in tablet manufacture. Such further components may be present asdiscrete particles, or may be components of the particles oftissue-reactive material and/or polymerisable and/or cross-linkablecomponent.

Nature of the Core

The principal functions of the core are to provide the sheet withstructural integrity and to provide a flexible substrate onto which thetissue-reactive formulation, in powder form, can be applied.

The core can be prepared using any suitable polymeric material orcombination of materials. The core may be biodegradable ornon-biodegradable, and should be biocompatible, ie should be capable ofapplication to tissues either within or external to the body withoutcausing any immunological or other adverse reaction.

Examples of polymeric materials that may be used for the core are:

-   Polymers or co-polymers based on a-hydroxy acids, such as    polylactide, polyglycolide; and also polycaprolactone and other    polylactones such as butyro- and valerolactone.

Other examples may include:

-   alginates (ie polymers based on alginic acid, the polysaccharide    obtained from seaweeds);-   polyhydroxyalkanoates;-   polyamides;-   polyethylene;-   propylene glycol;-   water-soluble glass fibre;-   starch;-   cellulose;-   collagen;-   pericardium;-   albumin;-   polyester;-   polyurethane;-   polyetheretherketone (PEEK);-   polypropylene; and-   polytetrafluoroethylene.

The core may be prepared by casting, spinning or foaming of a solutionof the polymeric material, or by moulding, weaving of filamentousmaterial, or slicing from a block of material. Appropriate techniquesfor the preparation of the core by such methods will be familiar tothose skilled in the art.

In preferred embodiments, the core is formed with a regular array ofapertures, for example in a square or hexagonal array. The apertures maybe formed during manufacture of the core or may be introduced after thecore is formed, for example by piercing.

Preferably, the apertures are between 50 μm and 2 mm in diameter andadjacent apertures are formed at a centre-to-centre separation ofbetween 100 μm and 5 mm. Preferably, the apertures account for between5% and 80% of the overall surface area of the core.

The core may have a thickness of from 0.005 to 5 mm.

Application of the Formulation to the Core

The formulation may be applied to just one side of the core. Morepreferably, however, the formulation is applied to both sides of thecore.

Where the core is apertured, application of the formulation to one orboth sides of the core causes the apertures to be filled with theformulation. In use, where the formulation is applied to both sides ofan apertured core, the formulation that is present in the perforationseffectively binds together the activated coatings on each side of thecore, encapsulating the core between the two layers of activatedcoating.

The preferred method for applying the formulation to the core involvesmechanically compressing (eg using a hydraulic press) a blend of thetissue reactive material and the polymerisable and/or cross-linkablecomponent, both in particulate form, onto one or both sides of the core.

The blend may be prepared by admixing particles of the tissue-reactivematerial with particles of the polymerisable andlor cross-linkablecomponent.

The coating formulation may include filler materials that may typicallyaccount for up to 50% by weight of the coating formulation. Examples ofsuch materials include cellulose derivatives (eg carboxymethylcellulose, hydroxypropyl methylcellulose, etc), polyethylene glycol,polyvinylpyrrolidone and other commonly used pharmaceutical excipients.

The thickness of the coating applied to one or both sides of the corewill typically be between 50 μm and 500 μm, more commonly from about 70μm to about 200 μm.

Optionally, a surface of the sheet that, in use, is not intended toadhere to tissue may be coated with a non-adhesive material. Mostpreferably, such a material is a synthetic polymer. Examples of suitablepolymers include polyethylene glycols, polylactide andpoly(lactide-co-glycolide). A sheet with such a non-adhesive coatingwill adhere only to the target tissue (to which the underside of thesheet is applied) and not to surrounding tissues (eg the pleural orperitoneal wall). The non-adhesive coating may include avisibly-absorbing chromophore to enable identification of the non-tissuecontacting surface of the sheet. An example of a suitable chromophore ismethylthioninium chloride.

The non-adhesive coating is preferably also formed with apertures. Insuch a case, the apertures may be formed in a similar array to theapertures in the core, with similar separations between apertures. Theapertures in the non-adhesive coating are, however, preferably somewhatsmaller than those in the core, eg with a diameter of between 50 μm and1 mm.

Physical Form of the Sheet

The sheet may typically have an overall thickness of from 0.05 to 10 mm,typically 0.05 to 2 mm, and more commonly 0.05 to 0.5 mm, eg about 200μm or 300 μm or 400 μm.

The sheet may be produced with, or subsequently cut to, dimensions offrom a few square millimetres up to several tens of square centimetres.

Therapeutic Applications of the Formulation and Sheet

The formulation and sheet according to the invention are suitable forapplication to both internal and external surfaces of the body, ie theymay be applied topically to the exterior of the body (eg to the skin) orto internal surfaces such as surfaces of internal organs exposed duringsurgical procedures, including conventional and minimally invasivesurgery.

The formulation and sheet according to the invention are particularlysuitable for surgical applications in the following areas:

-   Thoracic/cardiovascular-   General surgery-   ENT-   Urology-   Oral/maxillofacial-   Orthopaedic-   Neurological-   Gastroenterology-   Ophthalmology-   Gynaecology/obstetrics

Possible uses are described in more detail below.

Wound Healing

The degradable nature of the formulation and sheet mean that they maysupport and promote wound healing during both internal and topicalprocedures. Once the formulation and/or sheet begin to degrade,fibroblasts will move in and begin to deposit components of theextracellular matrix. The formulation and sheet can therefore be used asan internal or external dressing. In addition, factors such as growthfactors and cAMP that are known to promote the proliferation of skincells may be added to the formulation to assist in the healing process.The sheet may be designed to control the transmission of moisture andinfectious agents, and thus be useful particularly in the treatment ofburns.

Skin Closure

The formulation and sheet may be applied topically to promote woundclosure (as an alternative to sutures). This may have beneficial effectsin that it may reduce scarring, and the formulation and sheet may thusbe useful for cosmetic purposes during minor surgery (eg in Accident andEmergency Departments). The self-adhesive properties of the sheet makeit easy to apply quickly.

Hernia Repair

The sheet may be used to provide reinforcement in hernia repairprocedures. The self-adhesive attachment overcomes the potential issuesfaced by conventional surgical reinforcing mesh products, which requiresuturing or stapling in an already weakened area. The sheet for such aprocedure may be engineered to have short or long term durability,depending on the degree of tissue repair required. The sheet may also beable to withstand the application of staples.

Anastomosis

The formulation and self-adhesive sheet provide a means for rapidsealing of, and prevention of leaks in, joined tubular structures suchas blood vessels, and vascular and bladder grafts, and the GI tract. Theability of the sheet to support tissue repair may be of particular valueif used in nerve repair.

Sealing Large Areas of Tissue

The good sealing and handling properties of the formulation and sheet,combined with their self-adhesive properties and ability to cover alarge surface area, mean that they may be of particular use in sealingresected tissue surfaces—in particular those where diffuse bleeding isan issue (eg the liver). The sheet also provides an ideal support matrixfor tissue repair at such sites. This could also be applicable tolimiting leakage of cerebro-spinal fluid following neurological surgery.

Sealing Air Leaks

In addition to the patch properties described above, the high tensilestrength and good inherent elasticity of the formulation and sheet(after hydration and reaction of the tissue-reactive functional groups),make them particularly suitable for sealing air leaks in the lung,particularly following lung resection. Again, after effecting a seal,the sheet provides an ideal support matrix for tissue repair at suchsites.

Haemostasis

The formulation and sheet may be applied to a bleeding area, acting as aphysical barrier. The tissue-reactive material in the formulation andsheet may immobilise proteins and thereby promote haemostasis.

Therapeutic Agent Administration

Drugs and other therapeutic agents (including biologically active agentssuch as growth factors, and even cells and cellular components) may beadded to solution(s) used to form the components of the formulation andsheet, or covalently linked to components prior to their use in themanufacture of the formulation and sheet. Once the formulation or sheetis in place, following application to the desired site, the drug will beslowly released, either by diffusion or by engineering the formulationor sheet so that as it degrades over time the drug is released. The rateof release can be controlled by appropriate design of the formulationand sheet. The formulation and sheet may thus provide a means fordelivering a known amount of drug either systemically or to a preciselocus. The drug may be directly bound to a component of the formulation,or simply dispersed in the formulation.

Prevention of Post-Surgical Adhesions

Post-surgical adhesion, the formation of undesired connective tissuebetween adjacent tissues, is a serious problem which can give rise tomajor post-surgical complications. It is a particular problem in bowelsurgery where it can cause, for instance, twisting of the bowel, whichmay then necessitate further surgical intervention. The application ofsheet material having self-adhesive properties in accordance with theinvention to tissues exposed in a surgical procedure can be effective inpreventing post-surgical adhesions between that tissue and neighbouringtissues.

Minimally Invasive Procedures

The use of minimally invasive techniques for taking tissue samples bybiopsy, inserting devices, delivery of therapeutic agents and performingsurgical procedures is rapidly developing as an alternative choice totraditional “open” surgery. Minimally invasive procedures typicallyresult in less pain, scarring, quicker recovery time and fewerpost-operative complications for patients, as well as a reduction inhealth care costs. Procedures are undertaken using specially designedinstruments which are inserted through small keyhole-sized surgicalincisions. The formulation and sheet may be introduced into the body viaexisting and specially designed minimally invasive surgery instrumentsand trocar systems, and the sheet may be shaped or prepared to anappropriate size and configuration. The format of the formulation alsomay be modified to enable delivery of powders, tablets, pellets,tapes/strips/plegets and other 3-D matrices. The use of a self adhesiveformulation will significantly reduce the technical difficultiesassociated with manipulating, closing and repairing tissues where accessis restricted. In addition the sheet properties make them particularlysuitable for sealing leaks of air, blood or fluid or for delivery oftherapeutic agents.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention will now be described in greater detail, by way ofillustration only, with reference to the following Examples.

EXAMPLE 1 Synthesis of NHS-activated PVP-co-PAA

(a) Polymerisation of Acrylic Acid and N-vinyl-2-pyrrolidone

The polymer is formed via the polymerisation of monomers such asN-vinyl-2-pyrrolidone and acrylic acid, as shown in FIG. 3.

A number of methods may be used to initiate the polymerisation, such asfree radical, ionic (cationic or anionic), thermal, UV, redox etc. Freeradical polymerisation is the preferred polymerisation method and2-2′-azo-bis-isobutyrynitrile (AIBN) is the preferred initiator. TheAIBN decomposes into two radicals which can then attack thecarbon-carbon double bond in the vinylic monomer (acrylic acid) as shownin FIG. 4.

This will continue until termination of chain growth, via combination,disproportionation etc.

The reaction solvent may be N,N′-dimethylformamide, toluene, or anyother suitable solvent with a boiling point greater than 100° C. Tolueneis the currently preferred solvent.

A typical polymerisation method is as follows:

Solvent is charged to the reaction flask. Usually, around 5-10 ml ofsolvent per gram of monomer is sufficient. The flask is heated in an oilbath to a temperature sufficient for the generation of free radicalsfrom the chosen initiator. 80-85° C. is the optimum temperature whenusing AIBN as the initiator. Oxygen-free nitrogen is bubbled through thesolvent to remove any dissolved oxygen. Oxygen is also removed from themonomers in the same manner. The initiator is added to the solvent andallowed to dissolve. The monomers are added and the vessel closed. Anitrogen inlet and an escape needle may also be used.

The reaction may be allowed to stand for around 3-24 hours. The reactionmixture is cooled and the polymer is isolated from the solvent/polymersolution by precipitation in 5:1 hexane/isopropanol followed byfiltration. Successive washes with diethyl ether are required to removeall traces of polymerisation solvent from the polymer. After successivediethyl ether washes, the polymer is dried under reduced pressure toconstant weight.

Typical reaction conditions are shown in Table I:

TABLE I Monomer (g) N-vinyl-2- T Time Polydispersity Solvent (vol)acrylic acid pyrrolidone AIBN (g) (° C.) (hrs) Yield Mw Mn Index (Mw/Mn)Toluene (100 ml) 1.5 (20 mol %) 8.5 (80 mol %) 0.02 (0.125%) 80 3 — — —— Toluene (100 ml) 0.7 (10 mol %) 9.3 (90 mol %) 0.02 (0.125%) 80 3 54%80040 38800 2.0 Toluene (100 ml) 0.7 (10 mol %) 9.3 (90 mol %) 0.04(0.25%) 80 3 58% 74240 38340 1.9 DMF (100 ml) 0.7 (10 mol %) 9.3 (90 mol%) 0.02 (0.125% 80 3 62% 54000 25150 2.1 Toluene (100 ml) 0.5 (7.5 mol%) 9.5 (92.5 mol %) 0.02 (0.125%) 80 3 — — — — Toluene (100 ml) 0.35 (5mol %) 9.65 (95 mol %) 0.02 (0.125%) 80 3 — — — —(b) Reaction of PVP-co-PAA and N-hydroxysuccinimide in the Presence ofDicyclohexylcarbodiimide

NHS-activated PVP-co-PAA is formed from the reaction of PVP-co-PAA andN-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide (DCC)(FIG. 5).

10 g of PVP-co-PAA containing 0.094 moles of acrylic acid repeat unitsis dissolved in 50 ml of dried N,N′-dimethylformamide by stirring in adry 100 ml round bottomed flask. 0.01 moles of N-hydroxysuccinimide(1.15 g) is added to the polymer solution and is allowed to dissolve.

DCC (2.06 g) is melted in an oven at 60° C. and added to the polymersolution. This is left to stir at room temperature for at least 24hours. The formation of a white precipitate (dicyclohexylurea) isobserved. After 24 hours the precipitate is removed by filtration, andthe flask and filter washed with a small amount of dry DMF. The polymeris isolated by precipitation in 5:1 hexane/iso-propanol and filtration.The polymer is further purified by repeated washes with dry diethylether. The yield is between 50% and 70%.

EXAMPLE 2 Alternative Synthesis of NHS-activated PVP-co-PAA

(a) Polymerisation

400ml of dried toluene is heated to 80±2° C. in a round bottomed flaskusing an oil bath or isomantle. Oxygen is removed from the solvent bybubbling oxygen-free nitrogen through the toluene for at least 30minutes. 0.1 g (0.006 moles) of azo-iso-butyronitrile (AIBN) dissolvedin 2 ml of toluene is added to the reaction flask using a syringe,immediately followed by 45.02 g (0.406 moles) of 1-vinyl-2-pyrrolidoneand 7.02 g (0.092 moles) of acrylic acid. The reaction is left undernitrogen at 80±2° C. for 17 hours; the polymer is insoluble in tolueneand forms a white precipitate as the reaction proceeds. After 17 hours,a further 0.1 g (0.006 moles) of AIBN is added and the reaction is keptat 80±2° C. for one further hour to polymerise any remaining monomer.The polymer is isolated by pouring into 2000 ml of rapidly stirred 1:1hexane:diethyl ether and subsequent filtration using a 10-16 μm filter.The polymer is dissolved in 200 ml of N,N-dimethylformamide (DMF) andstirred for approximately 60 minutes before being filtered through a10-16 μm filter. The polymer is precipitated in approximately 2000 ml ofrapidly stirred 5:1 hexane:iso-propanol and isolated by filtration usinga 10-16 μm filter. All traces of DMF and toluene are removed by washingand filtration with 500 ml of diethyl ether three times. The polymer isdried for at least 72 hours at 60° C. in vacuo.

(b) NHS-esterification.

The acid content of the polymer is calculated by titration against 1.0MNaOH.

20 g of the polymer are dissolved in 160 ml of dry DMF in a 250 mlround-bottomed flask using a magnetic stirrer. 4.28 g (0.037 moles) ofNHS are added and allowed to dissolve. 7.67 g (0.037 moles) ofdicyclohexyldicarbodiimide are dissolved in 10 ml of dry DMF and addedto the polymer/NHS solution. The flask is sealed and the reactionstirred for 96 hours at room temperature. A DMF insoluble material,dicyclohexylurea, is formed as a reaction by product and this isapparent as a white precipitate present in the reaction solution. After96 hours the dicyclohexylurea is removed by filtration using a 10-16 μmfilter and the polymer isolated by precipitation using 1275 ml of 5:1hexane:iso-propanol. This is removed by filtration using a 10-16 μmfilter. The polymer is purified further by dissolving in 170 ml of DMFand precipitation in 1275 ml of 5:1 hexane:iso-propanol three furthertimes. After the final precipitation the polymer is washed by stirringrapidly in 170 ml of diethyl ether until a fine white powder isobtained. This is dried for at least 72 hours at 60° C. in vacuo.

EXAMPLE 3 Blending of NHS-activated PVP-co-PAA with Freeze-Dried Albumin

-   a) Powders of NHS-activated PVP₈₀-co-PAA₂₀ copolymers (ie copolymers    consisting of 80 mol % vinyl pyrrolidone-derived units and 20 mol %    acrylic acid-derived units) have been blended (in ratios of 1:1, 2:1    and 4:1) with freeze-dried porcine albumin (Sigma Aldrich;    previously buffered to pH 10.5).-   b) Powders of NHS-activated PVP₇₀-co-PAA₃₀ copolymers (70 mol %    vinyl pyrrolidone: 30mol % acrylic acid) have been blended (1:1)    with freeze-dried human albumin (Baxter human albumin solution (20%)    previously buffered to pH 10.5).-   c) Powders of NHS-activated PVP₇₀co-PAA₃₀ copolymers have been    blended (2:1) with freeze-dried porcine albumin (previously buffered    to pH 10.5).

EXAMPLE 4 Blending of NHS-activated PVP-co-PAA with Freeze-Dried Albuminand Application to Liver Tissue

a) Powders of NHS-activated PVP₈₀-co-PAA₂₀ have been blended (1:1) withfreeze-dried porcine albumin (Sigma Aldrich; previously buffered to pH10.5) and delivered onto moist liver tissue. The powder rehydratedrapidly (<5 minutes) yielding a gel that offers cohesive strength, inaddition to exhibiting strong adhesion to the underlying tissue surface.b) Powders of NHS-activated PVP₇₀-co-PAA₃₀ have been blended (1:1) withfreeze-dried human albumin (Baxter human albumin solution (20%)previously buffered to pH 10.5) and delivered onto moist liver tissue.The powder rehydrated rapidly (<5 minutes) yielding a gel that offerscohesive strength, in addition to exhibiting strong adhesion to theunderlying tissue surface.

EXAMPLE 5 Blending of NHS-activated PVP-co-PAA with Freeze-Dried PorcineAlbumin, Forming a Compressed Disc Followed by Application to LiverTissue

Powders of NHS-activated PVP₇₀-co-PAA₃₀ copolymers have been blended(2:1) with freeze dried porcine albumin (previously buffered to pH10.5), followed by compression into a thin (<2 mm thick) disc anddelivered onto moist liver tissue. The disc adheres immediately to theliver tissue and rehydrates gradually over an hour yielding a gel thatoffers cohesive strength, in addition to exhibiting strong adhesion tothe underlying tissue surface.

EXAMPLE 6 Blending of Excipients with Powdered NHS-activatedPVP₈₀-co-PAA₂₀ and Freeze-Dried Porcine Albumin Previously Buffered topH 10.5 (PSA)

Powders of NHS-activated PVP₈₀co-PAA₂₀ and PSA (1:1) have been blendedwith excipients such as hydroxypropyl cellulose, poly(vinyl pyrrolidone)and microcrystaline cellulose. The powdered mixture was compressed intoa disc with a thickness of less than 2 mm. These discs adheredimmediately to moist porcine liver tissue and rehydrated upon immersionin aqueous solution. After immersion in aqueous solution for 1 hour,they remained adhered to tissue as a crosslinked gel. Adhesion wasobtained with concentrations of NHS-activated PVP₈₀-co-PAA₂₀ and PSAfrom 11.5% to 50% w/w.

EXAMPLE 7 Schematic Representation of a Sheet According to the Invention

FIG. 6 shows (schematically and not to scale) the structure of a typicalsheet prepared in accordance with the invention. The sheet comprises acore in the form of a film 1 of poly(lactide-co-glycolide) (PLG) whichhas a regular array of apertures 5. Layers 2,3 of a tissue-reactiveformulation are pressed onto both sides of the film 1 such that thetissue-reactive formulation penetrates into, and fills, the apertures.Finally, a non-adhesive layer 4, again of PLG, is applied to one surfaceof the sheet. The non-adhesive layer 4 is also perforated, theperforations 6 being smaller than the apertures 5 in the core film 1.

The non-adhesive layer 4 may include a chromophore that gives thenon-adhesive surface a discernible colour, thereby identifying thatsurface (and hence indicating which side of the sheet is to be appliedto the tissue). Alternatively, the two sides of the sheet may bedistinguishable by virtue of a difference in reflectivity of the twosurfaces.

EXAMPLE 8 Preparation of Multilayer Sheet Formulation

This Example describes the preparation of a multilayer tissue-adhesivesheet having the structure illustrated schematically in Example 6. Thesheet comprises a PLG core, to both sides of which a particulate mixtureof NHS-activated PVP-co-PAA is applied. A PLG barrier layer is appliedto one side of the sheet.

8.1 Preparation of Poly (DL-lactide-co-glycolide) Core Film

The core film was prepared by casting from a 10 % w/w solution ofpoly(D,L-lactide-co-glycolide) (PLG) in dichloromethane. The core filmwas produced by spreading the 10% polymer solution using a 300 μm K Bar(R K Print Coat Instruments Ltd, Royston, UK) on silicon paper. A K Baris a device for accurately producing films of a specific thickness froma specific concentration solution. After drying, the thickness of thecore film was 30 μm.

8.2 Preparation of PLG Barrier Film

The barrier film was produced in an analogous manner to the core film,but using a 24 μm K Bar. After drying, the thickness of the barrier filmwas 3 μm.

7.3 Preparation of Perforations and Cutting Out ofpoly(DL-lactide-co-glycolide) Core and Barrier Films

The next step was to perforate the core and barrier films. This wascarried out using a heated perforating device in the form of a pressthat had been adapted for the purpose. The press was fitted with aheated plate, the underside of which was formed with a regular array ofpyramidal projections.

The perforations were created by applying the heated plate with pressureto the film. This softened the polymer film, which redistributed itselfaround the small pyramids. For perforating the core film, the heatedplate was set to 90° C. and pressed for 10 s. The barrier film wasperforated using a temperature of 90° C. for 5 s at a reduced pressingpressure. The perforation size for the core film was 1.3 mm with centreseparations of 2.5 mm and the barrier film perforation size was 0.5 mmwith centre separations of 2.5 mm. The perforated films were then cut tosize, which in this embodiment was a circle of 39.8 mm diameter. Due toredistribution of the film material during perforation, the filmthickness increased to 90 μm for the core film and 8-10 μm for thebarrier film.

8.4 Preparation of NHS-Activated PVP-co-PAA

NHS-activated PVP-co-PAA was prepared as described in Example 1.

8.5 Preparation of Freeze-Dried Buffered Human Serum Albumin

Freeze-dried buffered human serum albumin (FDBHSA) was prepared bymixing Human Albumin 20% solution with a pH10.5 sodium carbonate/sodiumphosphate solution. This solution was lyophilised, leaving powderedhuman albumin containing sodium carbonate and sodium phosphate evenlydistributed throughout.

The pH10.5 sodium carbonate/sodium phosphate solution was prepared by asfollows:

31.729 g (0.30 moles) of anhydrous sodium carbonate was weighed into asterile 250 ml glass bottle. Approximately 200 ml of water forinjections was added and the anhydrous sodium carbonate was dissolved bymixing on a roller mixer. Once the sodium carbonate had completelydissolved, the solution was poured into a 1000 ml volumetric flask andmade up to 1000 ml with water for injections.

A sodium phosphate solution was made by dissolving 3.560 g (0.03 moles)of sodium phosphate monobasic in approximately 50 ml of water forinjections in a glass bottle on a roller mixer. This was poured into a100 ml volumetric flask and made up to 1 00 ml using water forinjections.

The two solutions were mixed in the ratio 470 ml of sodium carbonatesolution to 90 ml of sodium phosphate solution. The two solutions werethoroughly mixed and the pH checked using a Mettler Toledo pH meter. ThepH of the solution should be in the range of pH 10-11. If the pH is toolow, sodium carbonate solution is added, and, if it is too high, sodiumphosphate solution is added, until the pH is within the desired range.

Human Albumin 20% solution was mixed 1:1 v/v with pH10.5 sodiumcarbonate/sodium phosphate in a glass bottle on a roller mixer foraround 30 minutes. When fully mixed, the Human albumin/sodiumcarbonate/phosphate solution was poured into porcelain dishes and frozenin a freezer at approximately −60° C. When the albumin solutions werecompletely frozen, they were transferred to the drying chamber of anEdwards Supermodulyo freeze-dryer. The chamber was sealed and the vacuumapplied. No heat was applied to the shelves and the albumin was left inthe freeze-dryer for at least 72 hours. The vacuum achieved was aminimum 10⁻¹ mbar.

When dry, the material was removed from the freeze-dryer and ground intoa fine powder using a pestle and mortar or electric mill.

8.6 Preparation of Mixed NHS-Activated PVP-co-PAA/FDBHSA Powders

The NHS-activated PVP-co-PAA and FDBHSA were ground together in a pestleand mortar until a fine powder was obtained. The ground powder was thenmixed on a roller mixer for 30 min prior to use to ensure the twocomponents were fully integrated.

8.7 Preparation of Multilayer Sheet

The final product was assembled in a Specac FT-IR 40 mm die bycompression between two pellets. Pieces of silicon paper were used toprevent the finished product sticking to the pellets.

With the first pellet in the die, a silicon paper disc was placed in thecavity and a 150 mg portion of the ground powder was sprinkled onto thesilicon paper. The powder was carefully manipulated with a spatula orplunger so that the powder evenly covered the entire base of the die.The 39.8 mm diameter perforated core film was placed on top of thepowder layer and firmly pressed so that so that the film was flat and incontact with the powder layer beneath it so that the powder occupied theperforations of the core film. A second 150 mg aliquot of powder wassprinkled onto the perforated PLG core film and again gently levelled.

The perforated barrier layer was placed on a second piece of siliconpaper and positioned on top of the second powder layer.

A second die pellet was introduced, and the assembled die placed intothe press and compressed to a pressure of 2 tonnes for 30 s. The finalproduct was then removed from between the pellets. The thickness of thefinal product was in the range 325-425 μm.

The side of the sheet to which the barrier layer is applied had a shinyappearance, and was hence distinguishable from the matt surface of thetissue-reactive side.

Example 9 Application of Sheet to Tissue

The tissue surface is prepared in accordance with conventional surgicaltechnique. The sheet is applied onto (and if necessary) around thetissue surface with moderate pressure to ensure satisfactory contact tothe tissue. Following application, the sheet may be hydrated with salinesolution.

Example 10 Measurement of Adhesive Strength

A Universal testing machine (UTM, Zwick/Roell BZ2,5) was used to testthe adhesive strength of test materials to freshly excised liver or lungtissue. Details of the testing procedure are summarised below.

A small section of tissue (4 cm×4 cm×1 cm (depth)) was prepared andmounted into a purpose-made holder at the base of the test machine. Thesurface of the tissue was sprayed with water. The test specimen (withsample holder attached to enable subsequent removal) was placed onto thetissue surface with a moderate force to ensure full contact. Thematerial was left on the tissue for 5 minutes and then wholly submergedin water for a further 5 minutes. Whilst holding the tissue in placeusing a suitable clamp the folded tip of the sample holder was Insertedin the grips of the UTM. The sample was positioned appropriately toensure that the sample was aligned with the grips. The grip was thenmoved at 180° from the test sample thereby removing the sample from thetissue. The UTM software (Zwick TestXpert ver 9.0) can be used tocalculate the energy of adhesion (mJ) of the test material. Adhesiontests were performed on powder compositions of differing compositions.

Table II shows the data obtained by testing compressed films as afunction of composition.

TABLE II Mean energy Test of adhesion medium Tissue reactiveCrosslinkable Ratio of (SD)/mJ Substrate material material components (n= 6) Porcine NHS-activated Human 4:1 1.3 (0.29) liver PVP₈₀-co- albuminPAA₂₀ Porcine NHS-activated Human 1:4 1.0 (0.43) liver PVP₈₀-co- albuminPAA₂₀ Porcine NHS-activated Human 1:1 1.0 (0.27) liver PVP₈₀-co- albuminPAA₂₀

The results listed in Table II demonstrate the adhesion performance ofthe co-powder formulations of NHS-activated PVP₈₀-co-PAA₂₀ andfreeze-dried human albumin.

In a further study, Table III illustrates the results of adhesiontesting of sheet formulations, (13 mm diameter circular discs) studiedas a function of the number of apertures in a PLG film with activeco-powders pressed into either side of the PLG film. The powdersutilised were NHS-activated PVP₈₀co-PAA₂₀ and human albumin in a 4:1ratio.

TABLE III No of apertures in Mean energy of adhesion Test medium 13 mmdiameter sheet (SD)/mJ (n = 6) Porcine liver 0 0.39 (0.14) Porcine liver10 0.75 (0.25) Porcine liver 20 1.14 (0.37)

The results listed in Table III demonstrate that the energy of adhesionof multi-layered sheets is proportional to the number of apertures inthe PLG inner film.

1. A tissue-adhesive formulation comprising a naturally occurring orsynthetic polymerisable and/or cross-linkable material in particulateform, the polymerisable and/or cross-linkable material is in admixturewith particulate material comprising tissue-reactive functional groups,wherein the polymerisable and/or cross-linkable material is albumin, andwherein the particulate material comprising tissue-reactive functionalgroups is the reaction product of poly(N-vinyl-2-pyrrolidone-co-acrylicacid) co-polymer and a reactant comprising a tissue-reactive functionalgroup wherein the tissue-reactive functional group is selected from thegroup consisting of imido ester, p-nitrophenyl carbonate,N-hydroxysuccinimide ester, epoxide, isocyanate, acrylate, vinylsulfone, orthopyridyl-disulfide, maleimide, aldehyde and iodoacetamide.2. A formulation according to claim 1, wherein the ratio by weight ofpolymerisable and/or cross-linkable material to material comprisingtissue-reactive functional groups is between 0.1:1 and 10:1.
 3. Aformulation according to claim 2, wherein the ratio by weight ofpolymerisable and/or cross-linkable material to material comprisingtissue-reactive functional groups is between 0.2:1 and 1:1.
 4. Aformulation according to claim 1, wherein the tissue-reactive functionalgroups are N-hydroxysuccinimide esters.
 5. A formulation according toclaim 1, wherein the poly (N-vinyl-2-pyrrolidone-co-acrylic acid)co-polymer has a molar ratio of acrylic acid-derived units less than0.60.
 6. A formulation according to claim 1, wherein the poly(N-vinyl-2-pyrrolidone-co-acrylic acid) co-polymer has a molar ratio ofacrylic acid-derived units between 0.025 and 0.25.
 7. A formulationaccording to claim 1, wherein the poly(N-vinyl-2-pyrrolidone-co-acrylicacid) co-polymer is derivatised with N-hydroxysuccinimide ester to formthe material comprising tissue-reactive functional groups.
 8. Aformulation according to claim 1, wherein the concentration of materialcomprising tissue-reactive functional groups in the formulation isbetween 10 and 50% w/w.
 9. A formulation according to claim 1, whereinthe albumin is porcine, bovine or human albumin.
 10. A formulationaccording to claim 1, wherein the polymerisable and/or cross-linkablematerial is buffered to a pH greater than
 7. 11. A formulation accordingto claim 1, further comprising one or more components selected from thegroup of structural polymers, surfactants, and plasticisers.
 12. Aformulation according to claim 1, wherein the particles that make up theformulation have a median size in the range 5 μm to 500 μm.
 13. Aformulation according to claim 1, which consists essentially of saidnaturally occurring or synthetic polymerisable and/or cross-linkablematerial in particulate form and said particulate material comprisingtissue-reactive groups.
 14. A sheet having a multilayer structure, saidstructure consisting of a core of a naturally occurring or syntheticpolymeric material, the core being coated on at least one side thereofwith a tissue-adhesive formulation according to claim
 1. 15. A sheetaccording to claim 14, wherein the naturally occurring or syntheticpolymeric material is selected from the group consisting of polymers orco-polymers based on α-hydroxy acids.
 16. A sheet according to claim 14,wherein the naturally occurring or synthetic polymeric material isselected from the group consisting of alginates, polyhydroxyalkanoates,polyamides, polyethylene, propylene glycol, water-soluble glass fibre,starch, cellulose, collagen, pericardium, albumin, polyester,polyurethane, potyetheretherketone, polypropylene andpolytetrafluoroethylene.
 17. A sheet according to claim 14, wherein thecore is apertured.
 18. A sheet according to claim 17, wherein the sheethas a regular array of apertures, and the apertures are between 50 μmand 2 mm in diameter and adjacent apertures are formed at acentre-to-centre separation of between 100 μm and 5 mm.
 19. A sheetaccording to claim 18, wherein the apertures account for between 5% and80% of the overall surface area of the core.
 20. A sheet according toclaim 14, wherein the core has a thickness of 0.005 to 5 mm.
 21. A sheetaccording to claim 14, wherein the tissue-adhesive formulation isapplied to the core by mechanically compressing a blend of materialcontaining tissue-reactive functional groups and polymerisable and/orcross-linkable material, both in particulate form, onto one or bothsides of the core.
 22. A sheet according to claim 14, wherein the coreis coated on both sides with the tissue-adhesive formulation.
 23. Asheet according to claim 14, wherein one surface of the sheet is coatedwith a non-adhesive material.
 24. A sheet according to claim 23, whereinthe non-adhesive material is selected from the group consisting ofpolyethylene glycols, polylactide and poly(lactide-co-glycolide).
 25. Asheet according to claim 24, wherein the non-adhesive coating includes avisibly-absorbing chromophore.
 26. A sheet according to claim 25,wherein the visibly-absorbing chromophore is methylthioninium chloride.27. A sheet according to claim 23, wherein the coating of non-adhesivematerial is apertured.
 28. A method of joining a tissue surface toanother tissue, or of sealing a tissue surface, which method comprisesapplying to the tissue surface a formulation according to claim
 1. 29. Amethod as claimed in claim 28, wherein the formulation is present on asheet having a multilayer structure consisting of a core formed of anaturally occurring or synthetic polymeric material, with theformulation being present as a coating on at least one side of the core.30. A method as claimed in claim 28, wherein the method is carried outto enhance wound healing, promote wound closure, provide reinforcementin hernia repair procedures, seal joint tubular structures, sealresected tissue sections, seal air leaks in lung tissue, promotehaemostasis, or prevent post-surgical adhesions.
 31. A method of joininga tissue surface to another tissue, or of sealing a tissue surface,which method comprises applying to the tissue surface a compositionaccording to claim
 14. 32. A method as claimed in claim 31, wherein themethod is carried out to enhance wound healing, promote wound closure,provide reinforcement in hernia repair procedures, seal joint tubularstructures, seal resected tissue sections, seal air leaks in lungtissue, promote haemostasis, or prevent post-surgical adhesions.